Open AccessResearch Induction of serine racemase expression and D-serine release from microglia by amyloid β-peptide Sheng-Zhou Wu1, Angela M Bodles2, Mandy M Porter2, W Sue T Griffin1,2
Trang 1Open Access
Research
Induction of serine racemase expression and D-serine release from microglia by amyloid β-peptide
Sheng-Zhou Wu1, Angela M Bodles2, Mandy M Porter2, W Sue T Griffin1,2,4, Anthony S Basile3 and Steven W Barger*1,2,4
Address: 1 Department of Neurobiology & Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA,
2 Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA, 3 DOV Pharmaceutical Inc., Hackensack, New Jersey, USA and 4 Geriatric Research Education and Clinical Center, Central Arkansas Veterans Healthcare System, Little Rock Arkansas, USA
Email: Sheng-Zhou Wu - wushengzhou@uams.edu; Angela M Bodles - bodlesangela@uams.edu; Mandy M Porter - portermandym@uams.edu;
W Sue T Griffin - griffinsuet@uams.edu; Anthony S Basile - abasile@dovpharm.com; Steven W Barger* - sbarger@uams.edu
* Corresponding author
Abstract
Background: Roles for excitotoxicity and inflammation in Alzheimer's disease have been
hypothesized Proinflammatory stimuli, including amyloid β-peptide (Aβ), elicit a release of
glutamate from microglia We tested the possibility that a coagonist at the NMDA class of
glutamate receptors, D-serine, could respond similarly
Methods: Cultured microglial cells were exposed to Aβ The culture medium was assayed for
levels of D-serine by HPLC and for effects on calcium and survival on primary cultures of rat
hippocampal neurons Microglial cell lysates were examined for the levels of mRNA and protein for
serine racemase, the enzyme that forms D-serine from L-serine The racemase mRNA was also
assayed in Alzheimer hippocampus and age-matched controls A microglial cell line was transfected
with a luciferase reporter construct driven by the putative regulatory region of human serine
racemase
Results: Conditioned medium from Aβ-treated microglia contained elevated levels of D-serine.
Bioassays of hippocampal neurons with the microglia-conditioned medium indicated that Aβ
elevated a NMDA receptor agonist that was sensitive to an antagonist of the D-serine/glycine site
(5,7-dicholorokynurenic acid; DCKA) and to enzymatic degradation of D-amino acids by D-amino
acid oxidase (DAAOx) In the microglia, Aβ elevated steady-state levels of dimeric serine racemase,
the apparent active form of the enzyme Promoter-reporter and mRNA analyses suggest that
serine racemase is transcriptionally induced by Aβ Finally, the levels of serine racemase mRNA
were elevated in Alzheimer's disease hippocampus, relative to age-matched controls
Conclusions: These data suggest that Aβ could contribute to neurodegeneration through
stimulating microglia to release cooperative excitatory amino acids, including D-serine
Alzheimer's disease (AD) involves neuronal cell loss and
reductions of synaptic density in specific brain regions
Some of the pathological signatures of AD implicate the
process of excitotoxicity For instance, glutamate receptors are altered in the AD brain [1], which also shows evidence
of activation of the calcium-triggered protease calpain [2]
Published: 20 April 2004
Journal of Neuroinflammation 2004, 1:2
Received: 22 March 2004 Accepted: 20 April 2004 This article is available from: http://www.jneuroinflammation.com/content/1/1/2
© 2004 Wu et al; licensee BioMed Central Ltd This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
Trang 2A glutamate receptor antagonist can reverse deficiencies in
synaptic transmission in a mouse model of AD [3]
Eleva-tions in glutamatergic stimulation may also contribute to
several other neurodegenerative conditions [4]
Most excitotoxic paradigms involve NMDA receptors,
complex ligand-gated calcium/sodium channels In
addi-tion to glutamate, the NMDA receptors require a
co-ago-nist at a second site Glycine has been the most extensively
studied ligand for this site However, D-serine shows an
approximately three-fold greater potency than glycine at
this site [5-7] D-serine satisfies several criteria for a
neu-rotransmitter or -modulator at NMDA receptors: selective
localization, controlled release, and physiological effect
Inactivation of D-serine by D-amino acid oxidase
(DAAOx) markedly reduces NMDA neurotransmission as
monitored by NO synthase activity and electrophysiology
in ex vivo cerebellar and hippocampal preparations [8].
Furthermore, injection of D-serine can modulate NMDA
receptor function in vivo [9,10] D-serine is generated from
the more prevalent L-serine by serine racemase (EC
5-1-1) Regulation of expression of serine racemase has not
been characterized, but under normal conditions the
enzyme is localized to the D-serine-containing
protoplas-mic astrocytes in areas of the brain rich in NMDA
recep-tors [11]
We recently reported that some of the derivatives of the
amyloid precursor protein (βAPP), including amyloid
β-peptide (Aβ), can stimulate glutamate release from
micro-glial cells [12] Aβ has been reported by many laboratories
to activate an inflammatory phenotype in microglia,
including the elevation of phagocytic activity, cytokine
expression, and production of NO and reactive oxygen
species [13-15] Because of the ability of D-serine to
coop-erate with glutamate in physiological and pathological
stimulation of the NMDA receptor, we tested whether
proinflammatory stimuli could influence the synthesis
and/or release of D-serine in microglia as well
Materials and methods
Materials
Aβ1–42 was purchased from Anaspec (San Jose CA)
Lyophilized peptide was dissolved in anhydrous dimethyl
sulfoxide at 2 mM, diluted with minimal essential
medium (Earle's salts) (MEM) to 150 µM and incubated
16–24 h at 37°C Recombinant sAPPα was produced and
purified as described previously [12] Lipopolysaccharide
(LPS) and 5,7-dicholorokynurenic acid (DCKA) were
from Sigma (St Louis MO) D-amino acid oxidase
(DAAOx) was from Worthington Biochemicals
(Lake-wood NJ); for heat-inactivation controls, DAAOx was
incubated for 15 min at 80°C The antibody against serine
racemase was from Becton-Dickinson/Transduction
Labo-ratories (Mississauga ON)
Cell culture
Primary microglia were obtained from mixed glial cul-tures generated from neonatal Sprague-Dawley rats as described previously [12] Briefly, cortical tissue was dis-sociated and plated in MEM supplemented to 10% with fetal bovine serum (FBS), 0.5 mM L-glutamine, and 10 µg/
mL gentamycin After 10–14 days, microglia were removed by vigorous lavage and plated into secondary culture A second lavage 30 min after secondary plating removed the astrocytes and oligodendrocytes; resulting secondary cultures were >95% microglia as determined by
staining with Griffonia simplicifolia isolectin B4 and glial
fibrillary acid protein (GFAP; exclusionary)
For RNA or protein harvest, cells were plated at 4 × 105/ dish in 35-mm dishes For collection of conditioned medium, cells were plated at 2 × 105/well in 24-well plates Cultures were changed to serum-free MEM before stimulation
Primary cultures of hippocampal neurons were estab-lished from E18 Sprague-Dawley rats as described previ-ously [12] Cultures were maintained in Neurobasal/B27 (Invitrogen) for 8–10 days before use in experiments The N9 mouse microglial cell line (courtesy of P Ricciardi-Castagnoli, Milan) and the HAPI rat microglial cell line (courtesy of J R Connor, Penn State U.) were maintained
in MEM/10% FBS
Measurement of D-serine
Reverse-phase HPLC was used to separate and detect D-serine in samples of conditioned medium, similar to the
methods of Hashimoto et al [16] For these experiments,
microglia were switched to MEM in which the concentra-tion of L-glutamine had been reduced to 10 µM; other-wise, the glutamine elution peak obscured that of D-serine Samples were derivatized by a 3:7 mixture of solu-tion A (30 mg/mL t-BOC-L-cysteine, 30 mg/mL o-phthal-dialdehyde in methanol):solution B (100 mM sodium tetraborate solution, pH 9.4) Resolution was achieved on two consecutive 4-µ NOVA-PAK C18 columns (Waters),
100 and 300 × 3.9 mm, respectively A linear gradient was established from 100% buffer A (0.1 M sodium acetate buffer, pH 6; 7% acetonitrile; 3% tetrahydrofuran) to 100% buffer B (0.1 M sodium acetate buffer, pH 6; 47% acetonitrile; 3% tetrahydrofuran) over 120 min at 0.8 mL/ min Fluorescence was monitored with 344 nm excitation and 443 nm emission
RT-PCR
Total RNA was isolated from microglial cultures and 1 µg was reverse-transcribed using the "Advantage RT-for-PCR" kit (Clontech); 1 µL of this product was used in PCR reac-tions with Clontech reagents Serine racemase mRNA lev-els were often so low as to require a two-step PCR to avoid
Trang 3nonlinear effects of reagent depletion; the first PCR was
10 cycles and the second utilized 10% of this product in a
25-cycle reaction Primers were designed to span an
intron/exon junction Mouse racemase; forward: 5'-GTT
ACT CAC AGC AGC GGA AAC C; reverse: 5'-GAG GGC
TCA GCA GCG TAT ACC (annealing at 61°C) Rat
race-mase; forward: 5'-TAG CGG GAC AAG GGA CAA TT;
reverse: 5'-TGC ATA CTT GAT TTC ATC TTC CGT G
(annealing at 61°C) Human racemase; forward: 5'-CTA
TCC ACC TCA CAC CAG TGC TAA C; reverse: 5'-ACA ATT
GAC GCT CCG TAG GCT (annealing temperature: 71°C)
Equivalency of input was confirmed by RT-PCR for
GAPDH as described previously [17]
Human subjects
Total mRNA was obtained from hippocampus of twelve
persons (38% males, ages 60–92) diagnosed with
Alzhe-imer's disease by CERAD criteria Nine (9) age-matched
controls (AMC) (87% male, ages 59–97) were free of
other neurological conditions and heart disease
Western blot analysis
Cell culture lysates were analyzed for serine racemase by
immunoblotting techniques described previously [18],
with the primary antibody diluted to 1:200 Blots were
digitized on a conventional scanner
Luciferase reporter assay
The serine racemase sequence representing nucleotides
1511 upstream of the start of translation was cloned from
the T98G human cell line; fidelity was confirmed by
sequencing This sequence was fused to the coding region
of firefly luciferase in pGL3-basic (Promega) to create
pGL3-RaceProm The pRL-TK vector (Promega) was used
as a cotransfected control for transfection efficiency and
cell survival For each 2-cm2 transfection well, 2 µL
Lipo-fectamine 2000 (Invitrogen) were mixed in MEM with
300 ng pGL3-RaceProm, 10 ng pRL-TK, and 690 ng
mis-cellaneous DNA, and this mixture was incubated at room
temperature for 20 min The mixture was applied to HAPI
cell cultures for 2 h, then removed by a medium change to
fresh MEM with or without agonists as indicated After an
additional 24 h, a lysate of each culture was assayed
sequentially for firefly luciferase and Renilla luciferase
activity with a commercial kit (Promega)
Calcium measurements
Primary neurons were assayed for intracellular ionic
cal-cium concentration ([Ca2+]i) as described previously [12]
Unless otherwise indicated, 800 nM tetrodotoxin was
present during measurements For DAAOx pretreatment
of conditioned media samples, the enzyme was dissolved
in the same buffer used during imaging, added to
condi-tioned media at a final concentration of 100 µg/mL
con-trol, and the mixture was incubated for 7 min at 37°C
Control incubations were performed with media diluted with an equal volume of the imaging buffer
Neuronal survival assay
Neurotoxicity was determined by measuring lactate dehy-drogenase (LDH) released into the culture medium using
a commercial kit (Sigma) Primary cultures from rat hip-pocampus were plated in 24-well plates, and glia were restricted by a two-day exposure to 1 µM cytosine arabino-side (AraC) Eight days after plating, neurons were treated with pharmacological agents and microglial conditioned medium Aliquots of culture medium were assayed for LDH 24–48 h later A survival index was generated wherein the lowest LDH reading from untreated condi-tioned medium was assigned a value of 100 (% survival) and the highest LDH reading from maximally lysed neu-rons was assigned a value of 0 (% survival) MTT assays were performed as described previously [19] For tests of the effect of DAAOx, microglia-conditioned medium was incubated as described above for calcium measurements
Results
As a first test of the role D-serine might play in Aβ-stimu-lated microglial neurotoxicity, we measured D-serine lev-els in microglia-conditioned medium Reverse-phase HPLC was performed on media samples, and conditions were determined under which D-serine could be quanti-fied Treatment of primary microglia with Aβ1–42 for 20–
24 h resulted in a large increase in D-serine in the medium (Fig 1) The maximal D-serine concentration varied between experiments, ranging from 115 to 660 µM LPS also evoked an increase in D-serine levels Neither Aβ nor LPS caused an elevation of glycine levels, which typically approximated the resting levels of D-serine (e.g., Fig 1B) MTT assays were also performed, excluding any artifacts of cell number or lysis (not shown)
D-serine is produced primarily by conversion from L-ser-ine by serL-ser-ine racemase This racemase is known to be
expressed in protoplasmic astrocytes in vivo To confirm its
expression in microglia, semi-quantitative RT-PCR was performed on mRNA isolated from several culture types Serine racemase expression was detected in cultures of pri-mary rat microglia and appeared to increase after activa-tion with Aβ (Fig 2A) We also surveyed microglial cell lines to exclude possible astrocyte contamination The HAPI rat microglial line contained serine racemase mRNA after treatment with Aβ (Fig 2B), as did the N9 mouse microglial line in the presence two other proinflamma-tory stimuli: sAPPα and LPS (Fig 2C) The mouse sequence was subcloned and sequenced to confirm identity
The presence of serine racemase mRNA in activated micro-glia raised the possibility that increases in expression of
Trang 4D-serine levels in microglial culture medium measured by HPLC
Figure 1
D-serine levels in microglial culture medium measured by HPLC A Chromatographic separation of amino acid
standards 1: L-Asp, Rt = 22 min; 2: L-Glu, Rt = 24.7'; 3: L-Ser, Rt = 26.2'; 4: D-Ser, Rt = 27.8'; 5: L-Gln, Rt = 29.3'; 6: Gly, Rt = 30.9'; 7: L-Arg, Rt = 33.2' B Chromatographic separation of actual microglia-conditioned medium C Primary microglia were
incubated 20 h with no addition (Con) or 15 µM Aβ1–42 Tracings are shown for aliquots of media from duplicates of each
treatment D D-serine values are represented as the mean ± SEM of triplicates (*p < 0.01), and results are representative of
three separate experiments
Trang 5this enzyme were responsible for the apparent elevations
of D-serine release by Aβ, so western blot analysis was
per-formed on cell lysates from primary microglia In both cell
lysates and positive control samples, the serine racemase
antibody detected monomeric protein (~37 kD) and an
apparent dimer (~74 kD) (Fig 3); specificity of the detec-tion was confirmed by a preabsorpdetec-tion control (Fig 3A) Such oligomers of the enzyme have been described recently and appear to include its soluble, active forms [20]; as reported in that study, we found the serine race-mase dimer to be insensitive to reducing agents Exposure
of primary microglia to Aβ had little or no effect on mon-omeric serine racemase but resulted in significantly higher levels of the apparent dimer (299% of control) (Fig 3B) Similar inductions were observed in the HAPI microglial cell line
To address the possibility of a transcriptional induction of serine racemase, a 1.5 kb sequence 5' to the luciferase cod-ing region was cloned from human genomic DNA This sequence was placed in the pGL3-basic plasmid for luci-ferase reporter assays HAPI microglial cells were trans-fected with this construct and treated with either Aβ or
Expression of serine racemase mRNA in activated microglia
Figure 2
Expression of serine racemase mRNA in activated
microglia Semi-quantitative RT-PCR was performed to
detect mRNA for serine racemase and GAPDH in microglial
cultures incubated 20 h in the absence (Con) or presence of
proinflammatory stimuli A Primary microglia treated with
15 µM Aβ1–42 [Densitometric analysis of racemase/GAPDH:
Con: 5.12 ± 0.64; Aβ: 9.78 ± 0.3 (p 0.005)] B HAPI
micro-glial cell line treated with 15 µM Aβ1–42 C N9 microglial cell
line treated with 300 ng/mL LPS or 10 nM sAPPα695
Induction of serine racemase by Aβ
Figure 3 Induction of serine racemase by Aβ Serine racemase
protein was detected by western blot analysis of lysates of
primary microglia A Microglial proteins were probed with
antibody that either had (+) or had not (-) been preabsorbed
to recombinant serine racemase The detection was inten-tionally overdeveloped to demonstrate nonspecific bands
dis-tinct from the monomer and unreducible dimer B Microglia
were incubated in triplicate for 12 h either with (+) or with-out (-) 15 µM Aβ1–42 Arrowhead designates monomer and arrow dimer Results are representative of three experi-ments Densitometry of the dimer in digitized images indi-cated a significant difference between treated and untreated
samples [cntrl: 139.97 ± 54.92, Aβ: 418.52 ± 74.37
(arbi-trary units); p < 0.02, unpaired Student's t-test]
Trang 6LPS After one day of treatment, luciferase levels indicated
an induction of the presumptive serine racemase
pro-moter by both stimuli (Fig 4)
Previous experiments demonstrated a release of glutamate
by microglia activated with sAPP and Aβ Useful in those
studies were bioassays in which hippocampal neurons
were monitored for intracellular ionic calcium
concentra-tion ([Ca2+]i) during application of conditioned medium
collected from control or activated microglia [12] As an
initial step to determine if proinflammatory activation of
microglia might evoke release of NMDA-R agonists other
than glutamate, we sought conditions suitable for
detecting ligands of the glycine/D-serine site of the NMDA
receptor With no other manipulations, application of
glutamate to hippocampal neurons elevated [Ca2+]i to
lev-els that were partially inhibited by a glycine/D-serine site
antagonist, 5,7-dicholorokynurenic (DCKA), suggesting
synaptic release of endogenous glycine To circumvent
this effect in bioassays of conditioned medium,
tetrodo-toxin (TTX) was employed This intervention resulted in a
[Ca2+]i response to glutamate that was smaller and
insensitive to DCKA (data not shown) Therefore, 800 nM TTX was included in subsequent bioassays of microglia conditioned medium Under these conditions, graded responses to D-serine could be detected at concentrations from 3–100 µM (data not shown)
Bioassays were performed on conditioned medium from primary microglia activated with either Aβ1–42 or LPS Hippocampal neurons responded to such media with a rapid increase in [Ca2+]i (Fig 5) Conditioned medium from Aβ-treated microglia evoked a modest response at a dilution of 1:100 into the imaging buffer; a 1:18 dilution elevated [Ca2+]i dramatically Medium from LPS-treated microglia had a similar effect (Fig 5B) By contrast, the conditioned medium from unactivated sister cultures showed no effect on neuronal [Ca2+]i at ratios up to 1:18 (Fig 5A) and evoked only a modest increase at 1:10 (Fig 5B) Acute treatment of neurons with equivalent amounts
of Aβ or LPS had no significant effect on [Ca2+]i DCKA (100 µM) reversed the [Ca2+]i response to microglia-con-ditioned medium The elevation was also sensitive to more general antagonists of the NMDA receptor (data not shown) As an independent test of the role of D-serine in the calcium responses evoked by microglia-conditioned medium, samples of media were incubated with D-amino acid oxidase (DAAOx) to remove D-serine; catalase was also added to the imaging buffer to obviate effects of
H2O2 produced by the DAAOx Treatment with DAAOx dramatically lowered the ability of microglia-conditioned medium to evoke a [Ca2+]i response (Fig 5C) Similar ele-vations of a DAAOx-sensitive NMDA agonist were observed in medium conditioned by the HAPI microglial cell line Several controls for the specificity of the DAAOx
treatments were performed (data not shown): i
exoge-nous glycine was able to overcome the effect of DAAOx, confirming independence from hydrogen peroxide or
similar artifacts of the DAAOx treatment; ii when samples
of conditioned media were treated with DAAOx that had been heat-inactivated, there was little difference from
untreated media samples; iii DAAOx was shown to be
specific for D-serine under the conditions of incubation
by tests in which the enzyme was incubated with [14 C]gly-cine or [3H]D-serine, subsequently analyzed by thin-layer chromatography
To explore the ramifications of D-serine release, we tested the influence of D-serine on neuronal health Primary hippocampal neurons were exposed to 1 or 3 µM D-ser-ine, and effects on metabolic activity were monitored by MTT assay the following day Treatment with 1 µM D-ser-ine lowered MTT values to 71.1% of control (±8.08), and
3 µM D-serine resulted in a value that was 11.54% of con-trol (±7.50) D-serine also generally potentiated the toxic-ity of low levels of glutamate (data not shown)
Responsiveness of serine racemase promoter to Aβ
Figure 4
Responsiveness of serine racemase promoter to Aβ
The human serine racemase upstream regulatory region was
cloned into a firefly luciferase reporter construct HAPI
microglial cells were cotransfected with this construct and a
vector encoding Renilla luciferase under control of a
constitu-tive promoter After transfection, the cells were treated in
serum-free medium with 0.3% DMSO ("Control"), 15 µM
Aβ1–42 or 100 ng/mL LPS Luciferase activity was measured
after 24 h and is represented as firefly luciferase signal,
rela-tive to Renilla luciferase signal in the same well (mean of
quadruplicates ± SEM; * p < 0.02; ** p < 0.001) Results are
representative of three separate experiments
Trang 7We next tested whether D-serine played a requisite role in the neurotoxicity exhibited by Aβ-treated microglia Pri-mary microglia were left untreated or were exposed to Aβ overnight Conditioned medium from these cells was applied to cultures of primary hippocampal neurons (Fig 6) A higher rate of LDH release was observed in the pres-ence of conditioned medium from Aβ-treated microglia compared to that obtained from untreated microglia As a control for the potential neurotoxicity of residual Aβ car-ried over with the conditioned medium, an aliquot of Aβ was diluted into culture medium in a cell culture dish lacking cells and incubated under identical conditions; treatment of neurons with medium thus prepared showed
no significant toxicity under the conditions of our assay (not shown) The neurotoxicity resulting from Aβ-acti-vated microglia was partially reversed by inclusion of 1 or
10 µM DCKA (Fig 6) Furthermore, pretreatment of the conditioned medium from Aβ-treated microglia with DAAOx also partially reversed its neurotoxicity Because DAAOx can generate hydrogen peroxide, a separate
Elevations in apparent D-serine detected by neuronal
bioassay
Figure 5
Elevations in apparent D-serine detected by neuronal
bioassay Primary hippocampal neurons were monitored for
[Ca2+]i during the application of conditioned medium (CM)
from microglia (for the period indicated by the lower bar)
DCKA (100 µM) was added as indicated by the bar thus
labeled A Microglia were cultured 20 h in the absence
(evenly dashed line) or presence of Aβ1–42 for 20 h CM from
Aβ-treated cultures was added to the neurons at either a
1:100 or 1:18 dilution B Microglia were cultured 20 h in the
absence (dashed line) or presence (solid line) of 300 ng/mL
LPS Both samples were added to neurons at a dilution of
1:10 C CM from Aβ-treated cultures was incubated with
DAAOx or a control buffer, then applied to neurons at a
1:18 dilution Similar results were obtained with conditioned
medium from LPS-stimulated microglia
Suppression of microglial neurotoxicity by DCKA and DAAOx
Figure 6 Suppression of microglial neurotoxicity by DCKA and DAAOx Primary microglia were treated for 24 h in
the absence (Con) or presence of 15 µM Aβ1–42 (Aβ) The
conditioned medium from these cultures was then diluted four-fold into the medium of primary hippocampal neuron cultures; neuronal viability was measured 24 h later by LDH release Some neuronal cultures received simultaneous appli-cation of 1 or 10 µM DCKA, and additional sets were exposed to microglia-conditioned medium that had been pre-treated with DAAOx Values represent the mean ± SEM
of triplicate determinations, and the results are representa-tive of three experiments (*p < 0.01 versus "no drug, +Aβ") Similar data were obtained using MTT reduction as an index
of viability
Trang 8treatment was tested utilizing catalase, but this did not
alter the effect of DAAOx (data not shown)
Based on the inductions by Aβ and other
proinflamma-tory stimuli, the levels of expression of serine racemase in
AD brain tissue were examined RT-PCR of mRNA isolated
from hippocampus of AD indicated a significant elevation
of serine racemase expression compared to age-matched
controls (AMC) (Fig 7) Within the AD group alone, the
female subjects showed a nonsignificant trend towards
higher levels than the male subjects The AD pool
con-tained a higher percentage of females, creating the
possibility that gender contributed to the difference
observed However, the difference between AD and AMC
was significant within males alone (ratios of
race-mase:GAPDH signals, AD: 1.62 ± 0.342; AMC: 0.447 ±
0.024; p < 0.05).
Discussion
The studies presented here document the capacity of
acti-vated microglia to express serine racemase and release
D-serine, thereby implicating D-serine as a contributor to
the neurotoxicity exhibited by inflammatory situations in
the CNS Microglia stimulated with Aβ or LPS released
D-serine, a potent NMDA receptor coagonist The
conditioned medium from such microglia elevated [Ca2+]i
in cultured hippocampal neurons in a manner that was largely reversed by D-serine/glycine-site antagonists, as well as more general antagonists of NMDA receptors Pre-treatment of the conditioned medium with DAAOx also blocked the effects on neuronal [Ca2+]i Aβ treatment ele-vated the steady-state levels of serine racemase mRNA and protein, suggesting that increased synthesis may be involved in the release of D-serine observed under these conditions Finally, a potential role for D-serine in Alzhe-imer's disease was further implicated by the observation that serine racemase mRNA is elevated in Alzheimer's brain tissue
D-serine has gained increased scrutiny as a NMDA
recep-tor agonist that may be more important than glycine in vivo, at least in specific regions or developmental stages.
However, the potential contribution of D-serine in excito-toxic pathologies has not received much attention Dam-age resulting from intracortical infusion of NMDA is attenuated by an inhibitor of poly-ADP ribose polymerase (PARP), and this neuroprotection was associated with a depression in the levels of D-serine but not glycine [21] Conventional wisdom held that D-serine/glycine sites on
NMDA receptors are typically saturated in vivo, making
elevations in their agonists irrelevant However, this idea has been refuted for over a decade now by observations of responsiveness to infused glycine [22] Similarly, applica-tions of D-serine have shown dramatic physiological effects [9,10,23,24] One set of results indicates that much
of the biological action of exogenous D-serine may come from stimulation of extrasynaptic NMDA receptors [8] To the extent that the actions of D-serine on NMDA receptors replicate those of glycine (perhaps, even more potently), the vast literature on exogenous glycine in excitotoxicity paradigms can be translated to D-serine But issues of production, release, uptake, and catabolism appear dis-tinct for these two glutamate co-agonists, making studies
of D-serine a distinct priority
Release of D-serine from astrocytes can be stimulated by non-NMDA, ionotropic glutamate receptor agonists [25] This fact has led to the hypothesis that the synaptic ele-ments of astrocytes may contribute to synaptic efficacy by participating in a positive-feedback loop whereby neuro-nal release of glutamate stimulates astrocytes to release D-serine and further amplify NMDA receptor activation [26] Recently, data were published consistent with the possibility that serine racemase is activated by direct bind-ing of calcium [20]; notably, AMPA/kainate receptor acti-vation elevates intracellular calcium levels in astrocytes [27] Therefore, a global release of glutamate – or, in fact, any strong calcium agonist – may lead to extrasynaptic release of D-serine, from both astrocytes and microglia Similar to its effects on neurons, Aβ can elevate [Ca2+]i in microglia [28], as can LPS [29] To wit, the degree of
Analysis of serine racemase mRNA in Alzheimer's disease
Figure 7
Analysis of serine racemase mRNA in Alzheimer's
disease Total RNA was isolated from hippocampus of AD
or age-matched control (AMC) brains.A Semi-quantitative
RT-PCR was performed with primers for serine racemase
and GAPDH B Densitometric analysis of PCR products is
represented graphically (*p < 0.02)
Trang 9elevation in D-serine released into medium was
surpris-ingly high given the changes in serine racemase protein
levels, suggesting that some of the Aβ-evoked increase in
D-serine release may have come from stimulation of
enzyme activity, in addition to expression levels Detailed
time-course analyses of protein levels and D-serine release
may provide some insight into this question
In the experimental paradigms applied here, the
presump-tive actions of D-serine in microglia-conditioned medium
were attenuated by DAAOx Under the normal conditions
of neurotransmission, glutamate concentrations at the
synapse are reduced primarily by astrocyte uptake [30]
The mechanisms controlling D-serine concentrations are
less clear; the relative contributions of degradation (e.g.,
by DAAOx), glial uptake, or diffusion out of the synaptic
cleft are topics of ongoing research There appears to be a
transporter for D-serine at the synapse [31], but it is
incompletely characterized Neuronal uptake, perhaps to
replenish presynaptic stores, would be consistent with the
finding that some pyramidal neurons in the cerebral
cor-tex and neurons in the nucleus of the trapezoid body
con-tain D-serine [32] Degradation of D-serine by DAAOx
produces hydrogen peroxide, creating potential for
addi-tional harm However, the concentrations of peroxide
pre-dicted from this reaction would be make a relatively
minor contribution to neuropathology compared to the
potent synergistic activation of NMDA receptors by
D-ser-ine and glutamate
A role for excitotoxicity in CNS inflammation is becoming
well established One of the first analyses of the relative
roles of various neurotoxins released by activated
micro-glia found that NMDA receptor antagonists were the most
efficacious neuroprotectants [33] Subsequently, Giulian
et al [34] described an excitotoxin released from
micro-glia exposed to amyloid plaques Excitotoxicity appears to
contribute to neuronal damage in more general models of
inflammation as well, such as intracerebroventricular LPS
infusion [35] Several studies have concluded that nitric
oxide (NO) mediates microglial neurotoxicity because
inducible NO synthase (iNOS) responds to
proinflamma-tory stimuli and general NOS inhibitors can be protective
in micoglia-neuron cocultures [36] However, most such
experiments cannot distinguish between NO generated by
microglia versus that generated by the neurons themselves
through classic excitotoxic mechanisms [37] When
corrected for Ki, inhibitors selective for neuronal NOS are
more potent protective agents than are iNOS-selective
compounds [12], suggesting that the primary neurotoxic
agents microglia produce are excitotoxins that activate
nNOS to produce NO within the neurons themselves
Previously, Li et al [38] showed that microglial cells
syn-thesize and release IL-1 in response to conditioned media
obtained from glutamate-stressed neurons The neurons respond with an increase in expression and processing of βAPP Secreted APP and Aβ can stimulate proinflamma-tory activation in microglia [13,18], including the release glutamate [12,39] These data are consistent with the plethora of evidence linking inflammatory mechanisms
to AD pathogenesis [40] Together with the potential for such stimuli to also trigger release of D-serine, these find-ings suggest that a vicious circle of inflammation and exci-totoxicity may be important in AD pathogenesis Excitotoxic events are a common aspect of many forms of neurodegeneration, even when they occur secondarily to ischemia or trauma, and considerable evidence suggests that excessive stimulation of glutamate receptors occurs in
AD [1-3]
Free D-serine concentrations are reported to be unaltered
in the Alzheimer brain [41,42] However, one study found
an elevation of overall serine levels in AD CSF per unit volume, but when normalized to protein concentration, the serine levels were similar between AD and controls [43], suggesting that elevated protein levels in AD CSF could confound analyses and interpretations Our initial analysis here indicates that there is an elevated steady-state level of serine racemase mRNA in AD hippocampus versus age-matched controls Nevertheless, the elevation
of D-serine itself might be expected to occur early in the disease progression; thus, any elevation might be difficult
to detect after the disease has progressed to its final stages Our semi-quantitative analysis showed that the mRNA for serine racemase was increased in AD brain nearly three-fold relative to age-matched controls It is possible that a portion of this difference can be accounted for by the hypothetical increase in numbers of astrocytes in AD However, a similar analysis of GFAP mRNA in AD reported levels to be only 57% higher in AD than in con-trols [44], and this effect includes an augmented expres-sion per cell [45] A recent microarray analysis of AD concluded that GFAP mRNA could not be compared to controls reliably due to variability across post-mortem interval, agonal state, etc [46]
In conclusion, Aβ and other AD-relevant proinflamma-tory stimuli are capable of stimulating release of D-serine from microglia Together with the release of glutamate evoked by similar conditions, a cooperative activation of NMDA receptors could be anticipated In addition to delineating details of the mechanisms by which CNS inflammation harms neuronal elements, this line of evi-dence may be relevant to the development of therapies If approaches targeting the general inflammatory system or glutamatergic neurotransmission are accompanied by unacceptable contraindications, a more specific interfer-ence with D-serine production or release may be more
Trang 10useful in AD and other neurodegenerative conditions For
this reason and others, it will be important to elucidate
the mechanisms controlling D-serine synthesis,
degrada-tion, and transport under normal and pathological
situations
Abbreviations
Aβ, amyloid β-peptide; DAAOx, D-amino acid oxidase;
DCKA, 5,7-dicholorokynurenate; HPLC, high pressure
liquid chromatography; LDH, lactate dehydrogenase; LPS,
lipopolysaccharide; MTT, methyltetrazolium; NMDA,
N-methyl D-aspartate; RT-PCR, reverse-transcriptase
polymerase chain reaction; sAPP, secreted amyloid
pre-cursor protein; TTX, tetrodotoxin
Competing interests
None declared
Authors' contributions
Author 1 (S-Z.W.) performed the calcium measurements,
neuronal survival experiments, DAAOx controls;
partici-pated in RT-PCR; cloned the racemase promoter and
per-formed the luciferase assays; and composed the first draft
of the manuscript Author 2 (A.M.B.) produced the
pri-mary microglial cultures, performed western blot analyses
and participated in the neuronal survival experiments
Author 3 (M.M.P.) was primarily responsible for RT-PCR
Author 4 (W.S.T.G.) provided the RNA samples from
characterized human cases and controls Author 5 (A.S.B.)
performed the HPLC measurements and participated in
the design of the study Author 6 (S.W.B.) conceived of the
study, participated in its design and coordination,
per-formed feasibility studies for the calcium measurements
and neuronal survival assays, and wrote the final draft of
the manuscript All authors read and approved the final
manuscript
Acknowledgements
Supported by the National Institute of Aging (R01AG17498 &
P01AG12411) We greatly appreciate Dr Camilo Rojas (Guilford
Pharma-ceuticals), who generously provided recombinant serine racemase for
pre-absorption antibody controls, as well as helpful discussions Richard Jones
also provided technical assistance.
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